A Fusion Protein of RGD4C and ß-Lactamase Has a Favorable Targeting Effect in Its Use in Antibody Directed Enzyme Prodrug Therapy.

Antibody directed enzyme prodrug therapy (ADEPT) utilizing ß-lactamase is a promising treatment strategy to enhance the therapeutic effect and safety of cytotoxic agents. In this method, a conjugate (antibody-ß-lactamase fusion protein) is employed to precisely activate nontoxic cephalosporin prodrugs at the tumor site. A major obstacle to the clinical translation of this method, however, is the low catalytic activity and high immunogenicity of the wild-type enzymes. To overcome this challenge, we fused a cyclic decapeptide (RGD4C) targeting to the integrin with a ß-lactamase variant with reduced immunogenicity which retains acceptable catalytic activity for prodrug hydrolysis. Here, we made a further investigation on its targeting effect and pharmacokinetic properties, the results demonstrated that the fusion protein retains a targeting effect on integrin positive cells and has acceptable pharmacokinetic characteristics, which benefits its use in ADEPT.

Radiation-sensitising effects of antennapedia proteins (ANTP)-SmacN7 on tumour cells.

The objective of this study was to investigate the underlying mechanisms behind the radiation-sensitising effects of the antennapedia proteins (ANTP)-smacN7 fusion protein on tumour cells. ANTP-SmacN7 fusion proteins were synthesised, and the ability of this fusion protein to penetrate cells was observed. Effects of radiation on the expression of X-linked inhibitor of apoptosis protein (XIAP) were detected by western blotting. The radiation-sensitising effects of ANTP-SmacN7 fusion proteins were observed by a clonogenic assay. The effects of drugs and radiation on tumour cell apoptosis were determined using Annexin V/FITC double staining. Changes in caspase-8, caspase-9 and caspase-3 were detected by western blot before and after ANTP-SmacN7 inhibition of XIAP. The ANTP-SmacN7 fusion protein could enter and accumulate in cells; in vitro XIAP expression of radiation-induced tumour cells was negatively correlated with tumour radiosensitivity. The ANTP-SmacN7 fusion protein promoted tumour cell apoptosis through the activation of caspase3. ANTP-SmacN7 fusion protein may reduce tumour cell radioresistance by inducing caspase3 activation.

Cytogenetic abnormalities in lymphocytes from victims exposed to cobalt-60 radiation.

The present study investigates cytogenetic damage in lymphocytes, derived from three victims who were unfortunately exposed to cobalt-60 (60Co) radiation (the 1999 accident occurred in a village in China's Henan province). Case A of the three victims was exposed to a higher dose of 60Co radiation than Cases B and C. The chromosomal aberrations, cytokinesis-block micronucleus (CBMN, the CBMN assay), and DNA double-strand breaks (DSBs, the comet assay) examined in this study are biomarkers for cytogenetic abnormalities. After the lymphocytes collected from the victims were cultured, the frequencies of dicentric chromosomes and rings (dic + r) and CBMN in the first mitotic division detected in the lymphocytes of Case A were found to be substantially higher than in Cases B and C. Similarly, the DNA-DSB level found in the peripheral blood collected from Case A was much higher than those of Cases B and C. These results suggest that an acutely enhanced induction of the 60Co-induced cytogenetic abnormality frequency in humans depends on the dose of 60Co radiation. This finding is supported by the data obtained using practical techniques to evaluate early lymphoid-tissue abnormalities induced after exposure to acute radiation.

Evaluation of the comet assay for assessing the dose-response relationship of DNA damage induced by ionizing radiation.

Dose- and time-response curves were combined to assess the potential of the comet assay in radiation biodosimetry. The neutral comet assay was used to detect DNA double-strand breaks in lymphocytes caused by γ-ray irradiation. A clear dose-response relationship with DNA double-strand breaks using the comet assay was found at different times after irradiation (p < 0.001). A time-response relationship was also found within 72 h after irradiation (p < 0.001). The curves for DNA double-strand breaks and DNA repair in vitro of human lymphocytes presented a nice model, and a smooth, three-dimensional plane model was obtained when the two curves were combined.

First-principles investigation of Ag-doped gold nanoclusters.

Gold nanoclusters have the tunable optical absorption property, and are promising for cancer cell imaging, photothermal therapy and radiotherapy. First-principle is a very powerful tool for design of novel materials. In the present work, structural properties, band gap engineering and tunable optical properties of Ag-doped gold clusters have been calculated using density functional theory. The electronic structure of a stable Au(20) cluster can be modulated by incorporating Ag, and the HOMO-LUMO gap of Au(20-) (n)Ag(n) clusters is modulated due to the incorporation of Ag electronic states in the HOMO and LUMO. Furthermore, the results of the imaginary part of the dielectric function indicate that the optical transition of gold clusters is concentration-dependent and the optical transition between HOMO and LUMO shifts to the low energy range as the Ag atom increases. These calculated results are helpful for the design of gold cluster-based biomaterials, and will be of interest in the fields of radiation medicine, biophysics and nanoscience.

ShRNA-mediated Ku80 gene silencing inhibits cell proliferation and sensitizes to gamma-radiation and mitomycin C-induced apoptosis in esophageal squamous cell carcinoma lines.

To investigate the effects of Ku80 depletion on cell growth and sensitization to gamma-radiation and MMC-induced apoptosis in esophageal squamous cell carcinoma lines. Six human carcinoma cell lines (LNcaP, K562, MDA-MB-231, MCF-7, EC9706, and K150) and normal HEK293 cell line were examined for basal levels of Ku80 protein by western blotting analysis. The suppression of Ku80 expression was performed using vector-based shRNA in EC9706 cells. Cell proliferation was determined with MTT assay and colony formation assay and tumorigenicity in a xenograft model in vitro and in vivo. Sensitivity of EC9706 cells treated with shRNA vector to gamma-radiation and MMC was determined with colony formation assay and MTT assay. The cell cycle distribution was determined by Flow cytometry. Apoptosis induced by gamma-radiation and MMC was analyzed using GENMED-TUNEL FACS kit. Ku80 showed higher basal levels in six carcinoma cell lines than in HEK293. The suppression of Ku80 expression decreased cellular proliferation, colony formation and inhibited tumorigenicity in a xenograft model. Furthermore, it sensitized apoptosis of the cancer cells induced by gamma-radiation and MMC. Ku80 plays an important role not only in tumorigenesis but also in radiation resistance and chemotherapy resistance in esophageal cancer cells. Hence Ku80 may serve as a promising therapeutic target, particularly for recurrent esophageal tumors.

Overexpression of Aurora-A kinase promotes tumor cell proliferation and inhibits apoptosis in esophageal squamous cell carcinoma cell line.

Aurora-A kinase, a serine/threonine protein kinase, is a potential oncogene. Amplification and overexpression of Aurora-A have been found in several types of human tumors, including esophageal squamous cell carcinoma (ESCC). It has been demonstrated that cells overexpressing Aurora-A are more resistant to cisplatin-induced apoptosis. However, the molecular mechanisms mediating these effects remain largely unknown. In this report, we showed that overexpression of Aurora-A through stable transfection of pEGFP-Aurora-A in human ESCC KYSE150 cells significantly promoted cell proliferation and inhibited cisplatin- or UV irradiation-induced apoptosis. Cleavages of caspase-3 and poly (ADP-ribose) polymerase (PARP) in Aurora-A overexpressing cells were substantially reduced after cisplatin or UV treatment. Furthermore, we found that silencing of endogenous Aurora-A kinase with siRNA substantially enhanced sensitivity to cisplatin- or UV-induced apoptosis in human ESCC EC9706 cells. In parallel, overexpression of Aurora-A potently upregulated the expression of Bcl-2. Moreover, the knockdown of Bcl-2 by siRNA abrogated the Aurora-A's effect on inhibiting apoptosis. Taken together, these data provide evidence that Aurora-A overexpression promoting cell proliferation and inhibiting apoptosis, suggesting a novel mechanism that is closely related to malignant phenotype and anti-cancer drugs resistance of ESCC cells.

[Detection of DNA injury and repair with single cell gel electrophoresis radiation in biodosimetry].

OBJECTIVE: To explore the relationship between DNA repair in vitro and in vivo after irradiation, and to describe the curves of DNA repair which can improve the accuracy of radiation dose estimation. METHODS: The DNA double-strand break in lymphocytes of human and mouse was detected using neutral single cell gel electrophoresis (SCGE) after radiation and the curves of DNA repair individually were estimated, which were compared later. RESULTS: Along with the time lapsing, the DNA repair of human peripheral blood and mice increased significantly and the residual damage decreased gradually, which showed significant time-effect relationship. The curve of DNA repair in vitro of human lymphocytes presented the same log model as that of mouse DNA repair in vivo. The curve showed as followed respectively: Mice: Y(TM) = 55.8256 - 10.792 lnX (R(2) = 0.629, P < 0.01) and Y(OTM) = 25.4173 - 4.5273 lnX (R(2) = 0.661, P < 0.01); Human: Y(TM) = 30.242 7 - 7.383 6 lnX (R(2) = 0.686, P < 0.01) and Y(OTM) = 17.9772 - 3.9125 lnX (R(2) = 0.752, P < 0.01). CONCLUSION: The curve of DNA repair in vitro of human lymphocytes could be considered in biodosimetry estimation because the process of DNA repair in vitro could display the repair level and speed of DNA double-strand break in vivo.

Experimental study on the new significant function domains of KCHIP1 protein.

Human K(v) channel interacting protein 1 (KCHIP1) is a new member of the neural calcium binding protein superfamily. Theoretically KCHIP1 has several calcium binding domains and two myristoylation sites. In this study, we demonstrated that the calcium binding domains and myristoylation sites were functional. The first, through running SDS-PAGE gel, we testified its ability of binding Ca(2+) with obvious discrepancy of the electrophoresis migrating rate after binding Ca(2+). Then, through the techniques of fused green fluorescence protein and site-directed mutagenesis, we demonstrated that wild type KCHIP1 protein accumulated in the secretory vesicles of Golgi body. In contrast, its two mutated forms without myristoylation sites accumulated throughout the whole cytoplasm. These observations indicate that KCHIP1 protein has a myristoylation motif mediating the interaction between KCHIP1 protein and membrane.

Increased miRNA-22 expression sensitizes esophageal squamous cell carcinoma to irradiation.

miRNA-22 was previously reported to be a tumor suppressor. The aim of this study was to explore the expression and function of miRNA-22 in esophageal squamous cell carcinoma (ESCC). Expression of miRNA-22 in 100 ESCC tissues was examined by q-PCR. The correlation between miRNA-22 level and clinicopathological features was analyzed using SPSS16.0 statistical software. Moreover, the effect of miRNA-22 expression on radiosensitivity of ESCC cells was examined. miRNA-22 expression decreased in ESCC tissues, and statistical analyses showed that the expression of miRNA-22 was associated with the stage of clinical classification. No correlation was found between miRNA-22 expression and the overall survival of ESCC patients. However, significant positive correlation was found between miRNA-22 expression and the survival of patients who received radiotherapy (P < 0.05). Increased expression of miRNA-22 sensitized ESCC cells to γ-ray radiation and promoted the apoptosis of ESCC cells induced by γ-ray radiation. Increased expression level of miRNA-22 had effects on Rad51 expression after irradiation. These results demonstrate for the first time that decreased miRNA-22 expression correlates with increased radiotherapy resistance of ESCC, and that this effect is mediated, at least in part, by the Rad51 pathway.

Effect of radiation on the Notch signaling pathway in osteoblasts.

Notch signaling has been shown to be important in osteoblast differentiation. Therapeutic radiation has been shown to alter the skeletal system, yet little information is available on the changes in Notch signaling in irradiated osteoblasts. The purpose of this study was to analyze the effect of radiation therapy with 2 and 4 Gy on Notch signaling in osteoblasts. In order to assess the radiation damage on osteoblast differentiation, total RNA and protein were collected three days after exposure to radiation. The effects of radiation on Notch signaling at the early and terminal stages of osteoblastic MC3T3-E1 cell differentiation was analyzed by qRT-PCR and western blot analysis. Our study applied a previously established method to induce MC3T3-E1 cell differentiation into osteoblasts and osteoblast precursors. Our results showed that the expression of Notch receptors (Notch1-4), ligands (Jagged1, Jagged2 and Delta1), target of Notch signaling (Hes1) and markers (ALP, M-CSF, RANKL and OPG) were altered following 2 and 4 Gy of irradiation. The present research did not indicate a strong relationship between Notch1 regulation and suppression of osteoblast differentiation. We found Hes1 may play a role in the radiation effect on osteoblast differentiation. Our results indicate that radiated osteoblast precursors and osteoblasts promoted osteoclast differentiation and proliferation.

Effect of radiation on the expression of osteoclast marker genes in RAW264.7 cells.

Cancer radiation therapy can cause skeletal complications, such as osteopenia and osteoporosis. To understand the mechanism responsible for the skeletal complications, the expression profiles of osteoclast marker genes in RAW264.7 cells were observed. Osteoclast formation was established by RAW264.7 cells that were treated with the receptor activator of nuclear factor (NF)-κB ligand (RANKL) and detected using immunochemistry and morphological observations. Quantitative real-time polymerase chain reaction was used to assess the expression of a panel of osteoclast markers, including the receptor activator of NF-κB (RANK), tartrate-resistant acid phosphatase (TRAP), integrin ß3 and the calcitonin receptor (CTR). RANKL-induced osteoclasts were TRAP-positive and multinucleated, and displayed a distinct morphology. RANKL-induced osteoclast precursor cells had increased TRAP and RANK expression and decreased CTR expression compared to the control cells not treated with RANKL. RAW264.7 cells irradiated with 2-Gy γ-rays had upregulated integrin ß3 and RANK expression and downregulated CTR expression compared to the control RAW264.7 cells. The effect of radiation on RANKL-induced osteoclast differentiation enhanced the expression of CTR and inhibited the expression of RANK and TRAP. Therefore, radiation damage from 2-Gy γ-rays can promote the activities of osteoclast precursor cells, but not those of osteoclasts.

In vivo renal clearance, biodistribution, toxicity of gold nanoclusters.

Gold nanoparticles have shown great prospective in cancer diagnosis and therapy, but they can not be metabolized and prefer to accumulate in liver and spleen due to their large size. The gold nanoclusters with small size can penetrate kidney tissue and have promise to decrease in vivo toxicity by renal clearance. In this work, we explore the in vivo renal clearance, biodistribution, and toxicity responses of the BSA- and GSH-protected gold nanoclusters for 24 h and 28 days. The BSA-protected gold nanoclusters have low-efficient renal clearance and only 1% of gold can be cleared, but the GSH-protected gold nanoclusters have high-efficient renal clearance and 36% of gold can be cleared after 24 h. The biodistribution further reveals that 94% of gold can be metabolized for the GSH-protected nanoclusters, but only less than 5% of gold can be metabolized for the BSA-protected nanoclusters after 28 days. Both of the GSH- and BSA-protected gold nanoclusters cause acute infection, inflammation, and kidney function damage after 24 h, but these toxicity responses for the GSH-protected gold nanoclusters can be eliminated after 28 days. Immune system can also be affected by the two kinds of gold nanoclusters, but the immune response for the GSH-protected gold nanoclusters can also be recovered after 28 days. These findings show that the GSH-protected gold nanoclusters have small size and can be metabolized by renal clearance and thus the toxicity can be significantly decreased. The BSA-protected gold nanoclusters, however, can form large compounds and further accumulate in liver and spleen which can cause irreparable toxicity response. Therefore, the GSH-protected gold nanoclusters have great potential for in vivo imaging and therapy, and the BSA-protected gold nanoclusters can be used as the agent of liver cancer therapy.

Methotrexate-mediated inhibition of RAD51 expression and homologous recombination in cancer cells.

BACKGROUND: Methotrexate is an inhibitor of folic acid metabolism. Homologous recombination is one of the most important ways to repair double-stranded breaks in DNA and influence the radio- and chemosensitivity of tumor cells. But the relationship between methotrexate and homologous recombination repair has not been elucidated. METHODS: Induction of double-strand breaks by methotrexate in HOS cells is assessed by the neutral comet assay. Inhibition of subnuclear repair foci by methotrexate is measured by immunofluorescence. Western blot and quantitative real-time PCR are conducted to detect whether methotrexate affects the expression level of genes involved in homologous recombination. In addition, we used a pCMV3xnls-I-SceI construct to determine whether methotrexate directly inhibits the process of homologous recombinational repair in cells, and the sensitivity to methotrexate in the Ku80-deficient cells is detected using clonogenic survival assays. RESULTS: The result showed that methotrexate can regulate the repair of DNA double-strand breaks after radiation exposure, and methotrexate inhibition caused the complete inhibition of subnuclear repair foci in response to ionizing radiation. Mechanistic investigation revealed that methotrexate led to a significant reduction in the transcription of RAD51 genes. Treatment with methotrexate resulted in a decreased ability to perform homology-directed repair of I-SceI-induced chromosome breaks. In addition, enhancement of cell death was observed in Ku mutant cells compared to wild-type cells. CONCLUSIONS: These results demonstrate that methotrexate can affect homologous recombination repair of DNA double-strand breaks by controlling the expression of homologous recombination-related genes and suppressing the proper assembly of homologous recombination-directed subnuclear foci.

Knockdown of Rad51 expression induces radiation- and chemo-sensitivity in osteosarcoma cells.

Osteosarcoma is the common primary bone malignancy in children and young adults in Eastern countries. Resistance to ionizing radiation (IR) or drugs is an underlying mechanism contributing to the failure of therapy in these patients. Rad51 is the key protein of DNA homologous recombination repair. Although high expression of Rad51 is associated with enhanced resistance to DNA damage induced by chemicals and/or ionizing radiation, the relevance of Rad51 expression in osteosarcoma and its relationship with IR sensitivity and chemo-resistance is not well understood. In this study, we elucidated the possibility of using Rad51 in the treatment of human osteosarcoma in vitro. Changes in chemo- and radiation sensitivity in cultured osteosarcoma cells occurred after suppression of Rad51 expression, using a plasmid vector-mediated short hairpin RNA (shRNA) expression system. The suppression of Rad51 correlated with cell cycle arrest in the G2 phase and inhibited tumor cell proliferation. Our results suggest that Rad51 expression levels might play an important role in radiation- and chemo-sensitivity of human osteosarcoma.

Size-dependent in vivo toxicity of PEG-coated gold nanoparticles.

BACKGROUND: Gold nanoparticle toxicity research is currently leading towards the in vivo experiment. Most toxicology data show that the surface chemistry and physical dimensions of gold nanoparticles play an important role in toxicity. Here, we present the in vivo toxicity of 5, 10, 30, and 60 nm PEG-coated gold nanoparticles in mice. METHODS: Animal survival, weight, hematology, morphology, organ index, and biochemistry were characterized at a concentration of 4000 µg/kg over 28 days. RESULTS: The PEG-coated gold particles did not cause an obvious decrease in body weight or appreciable toxicity even after their breakdown in vivo. Biodistribution results show that 5 nm and 10 nm particles accumulated in the liver and that 30 nm particles accumulated in the spleen, while the 60 nm particles did not accumulate to an appreciable extent in either organ. Transmission electron microscopic observations showed that the 5, 10, 30, and 60 nm particles located in the blood and bone marrow cells, and that the 5 and 60 nm particles aggregated preferentially in the blood cells. The increase in spleen index and thymus index shows that the immune system can be affected by these small nanoparticles. The 10 nm gold particles induced an increase in white blood cells, while the 5 nm and 30 nm particles induced a decrease in white blood cells and red blood cells. The biochemistry results show that the 10 nm and 60 nm PEG-coated gold nanoparticles caused a significant increase in alanine transaminase and aspartate transaminase levels, indicating slight damage to the liver. CONCLUSION: The toxicity of PEG-coated gold particles is complex, and it cannot be concluded that the smaller particles have greater toxicity. The toxicity of the 10 nm and 60 nm particles was obviously higher than that of the 5 nm and 30 nm particles. The metabolism of these particles and protection of the liver will be more important issues for medical applications of gold-based nanomaterials in future.

Expression and function of miRNA in postoperative radiotherapy sensitive and resistant patients of non-small cell lung cancer.

PURPOSE: To investigate the different miRNA expression profiles of postoperative radiotherapy sensitive and resistant patients of non-small cell lung cancer, explore their potential role and find some radio-sensitivity markers. MATERIALS AND METHODS: Thirty non-small cell lung cancer patients who have been treated by postoperative radiotherapy were selected and were divided into radiotherapy sensitive group and resistant group according to overall survival and local or distant recurrence rate. Expression profile of miRNA in these two groups was detected by a microarray assay and the results were validated by quantitative RT-PCR and Northern blot. At the molecular level, the effect of one differently expressed miRNA (miR-126) on the growth and apoptosis of SK-MES-1 cells induced by irradiation was examined. RESULTS: Comparing with resistant patients, five miRNAs (miRNA-126, miRNA-let-7a, miRNA-495, miRNA-451 and miRNA-128b) were significantly upregulated and seven miRNAs (miRNA-130a, miRNA-106b, miRNA-19b, miRNA-22, miRNA-15b, miRNA-17-5p and miRNA-21) were greatly downregulated in radiotherapy sensitive group. Overexpression of miRNA-126 inhibited the growth of SK-MES-1 cells and promoted its apoptosis induced by irradiation. The expression level of p-Akt decreased in miRNA-126 overexpression group. After treating with phosphoinositidyl-3 kinase (PI3K) constitutively activator (IGF-1) and inhibitor (LY294002), miRNA-126 overexpression had no significant effects on the apoptosis of SK-MES-1 cells. CONCLUSION: We found 12 differently expressed miRNAs in the radiotherapy sensitive and resistant non-small cell lung cancer samples. Moreover, our results showed miRNA-126 promoted non-small cell lung cancer cells apoptosis induced by irradiation through the PI3K-Akt pathway.

Construction, expression and functional characterization of the ß-lactamase with αv integrin ligands.

Antibody-directed enzyme prodrug therapy (ADEPT) delivers chemotherapeutic agents at high concentration to tumor tissues while minimizing systemic drug exposure. ß-Lactamases are particularly useful enzymes for ADEPT systems due to their unique substrate specificity, which allows the activation of a variety of lactam-based prodrugs with minimal interference from mammalian enzymes. This study used integrin α(v)ß(3) as a target for tumor-specific delivery of ß-Lactamase. ß-Lactamase was fused with ACDCRGDCFCG peptide (RGD4C) by recombinant DNA technology. Likewise, this study cloned a fused cDNA and successfully expressed active recombinant protein in E. coli purified with Ni-NTA resin. After purification, ß-Lactamase moiety showed the expected size of 42 kDa on Tricine-SDS-PAGE, and was further confirmed by Western blotting. Based on flow cytometric analysis, the purified protein was found to be active for specificity in breast cancer cell line, MCF-7, which supports the utility of the protein as an agent for ADEPT.

Toxicologic effects of gold nanoparticles in vivo by different administration routes.

Gold nanoparticles have potential applications in biomedicine, but one of the important concerns is about their safety. Most toxicology data are derived from in vitro studies and may not reflect in vivo responses. Here, an animal toxicity study of 13.5 nm gold nanoparticles in mice is presented. Animal survival, weight, hematology, morphology, and organ index are characterized at different concentrations (137.5-2200 µg/kg) over 14-28 days. The results show that low concentrations of gold nanoparticles do not cause an obvious decrease in body weight or appreciable toxicity, even after their breakdown in vivo. High concentrations of gold nanoparticles induced decreases in body weight, red blood cells, and hematocrit. It was also found that gold nanoparticles administered orally caused significant decreases in body weight, spleen index, and red blood cells. Of the three administration routes, the oral and intraperitoneal routes showed the highest toxicity, and the tail vein injection showed the lowest toxicity. Combining the results of all of these studies, we suggest that targeted gold nanopartices by tail vein injection may be suitable for enhancement of radiotherapy, photothermal therapy, and related medical diagnostic procedures.